Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, 8093, Switzerland.
Rapid Commun Mass Spectrom. 2019 Dec 30;33(24):1855-1860. doi: 10.1002/rcm.8546.
Ferritin is an iron storage protein assembly, usually formed by a 24-subunit protein shell and an iron core. The ferritin shell has been well studied using various structural biology tools such as X-ray diffraction and cryo-electron microscopy, whereas the iron status of ferritin is less studied and no well-established method exists for characterizing the distribution of the iron loading of ferritin. Recent advances in mass spectrometry (MS) have expanded the observable m/z range, making the measurement of ferritin possible with MS. In this study, matrix-assisted laser desorption ionization (MALDI)-MS was employed to quantify the iron content of ferritin.
The iron content of ferritin was quantified using a MALDI-MS system coupled with a commercially available ion conversions dynode high-mass detector. IgG1 antibody and its aggregates were used as external mass calibrants. The stability of HoloFt and ApoFt was also assessed in this study under different conditions, including various buffer pH, crosslinking agents and MALDI laser intensities.
The differences in peak width of HoloFt, ApoFt and IgG1 indicate the existence of mineral adducts in both HoloFt and ApoFt, and the mineral loading is heterogeneous among the HoloFt and ApoFt population. An average of 2773 ± 1584 iron atoms were determined for a commercial HoloFt sample. The iron core inside the ferritin complex is shown to stabilize and maintain the intact globular complex structure of ferritin.
This work introduces a MALDI-MS-based workflow for characterizing the ferritin iron loading pattern, which is meaningful for clinical analysis of iron deficiency/overload. In addition, the stability of ferritin is examined under various conditions, providing a guideline for further method development related to ferritin complex.
铁蛋白是一种铁储存蛋白组装体，通常由 24 个亚基蛋白壳和一个铁核心组成。铁蛋白壳已经通过各种结构生物学工具进行了很好的研究，如 X 射线衍射和冷冻电子显微镜，而铁蛋白的铁状态研究较少，也没有建立用于表征铁蛋白中铁负载分布的成熟方法。质谱（MS）的最新进展扩大了可观察的 m/z 范围，使得使用 MS 测量铁蛋白成为可能。在这项研究中，基质辅助激光解吸电离（MALDI）-MS 被用于定量铁蛋白的铁含量。
使用与市售离子转换 dynode 高质量检测器耦合的 MALDI-MS 系统定量铁蛋白的铁含量。IgG1 抗体及其聚集体被用作外部质量校准标准。在这项研究中，还评估了 HoloFt 和 ApoFt 在不同条件下的稳定性，包括不同的缓冲 pH 值、交联剂和 MALDI 激光强度。
HoloFt、ApoFt 和 IgG1 的峰宽差异表明 HoloFt 和 ApoFt 中都存在矿物质加合物，并且 HoloFt 和 ApoFt 群体中的矿物质负载不均匀。商业 HoloFt 样品的平均铁原子数为 2773±1584 个。铁蛋白复合物内部的铁核稳定并维持铁蛋白完整的球状复合物结构。
这项工作介绍了一种基于 MALDI-MS 的工作流程，用于表征铁蛋白的铁负载模式，这对临床缺铁/过载分析具有重要意义。此外，还在各种条件下检查了铁蛋白的稳定性，为进一步开发与铁蛋白复合物相关的方法提供了指导。
